Project Summary We have uncovered a new pathway in CD11c+ dendritic cells (DC) that controls T helper cell differentiation into the Th17 and Th2 lineages. The pathway involves cyclic AMP (cAMP) and protein kinase A (PKA), and plays a key role in the differential induction of immune defense and immunopathology at mucosal sites. In this project, we will address the underlying mechanisms by focusing on signaling by the heterotrimeric (???) GTP binding proteins, Gas (encoded by Gnas) and Gai (encode by Gnai2), which stimulate and inhibit cAMP synthesis, respectively. We have generated two powerful new physiologic models to explore cAMP functions in mucosal immune regulation, CD11c-specific conditional gene-targeted mice for Gnas (Gnas?CD11c, low cAMP) and Gai (Gnai2?CD11c, high cAMP). We discovered that Gnas?CD11c mice are biased toward Th2, whereas Gnai2?CD11c mice are prone to Th17, and that the two mouse strains display distinct mucosal immune abnormalities. These findings have led us to hypothesize that: I) Differential cAMP production in CD11c+ DC creates a pro-Th17 (high cAMP) or pro-Th2 (low cAMP) DC phenotypes; II) The imbalance of cAMP in the absence of Gas vs. Gai in DC results in differential immune reactivity in different mucosal organs; and III) Differential cAMP regulation in DC can be exploited pharmacologically as an innovative immune-modulatory strategy to treat Th17- and Th2-related pathologies and their related infections. We have assembled a multi- disciplinary team with complementary expertise to test these hypotheses in four Specific Aims. In Specific Aim 1 (SA1), we will assess the impact of Gas- and Gai-dependent signaling in DC on constitutive Th bias at different mucosal and systemic sites, and on spontaneous mucosal immunopathology. We will then evaluate the role of Gas and Gai on classical DC1 (cDC1) and cDC2 development and function. SA2 will explore the function of Gas and Gai in DC in controlling intestinal inflammation phenotypes in complementary models of colitis. SA3 will determine the importance of Gas and Gai in DC in antimicrobial host defense in the intestine, while SA4 will define Gas- and Gai- dependent mediators of DC-controlled Th17/Th2 polarization. Taken together, our discoveries in newly constructed Gnas?CD11c and Gnai2?CD11c mouse models have led to exciting and innovative hypotheses on the role of cAMP signaling in DC in orchestrating mucosal immune decisions, which will be systematically tested in this proposal. The results will yield new insight into the mechanisms of cAMP-dependent immune defense and immunopathology at mucosal sites. The findings will also form the basis for innovative immunotherapeutic strategies to target cAMP functions in DC. The project has a high likelihood of advancing basic immunological research, and addressing an unmet medical need in modulating destructive mucosal immune responses and in promoting protective responses against enteric pathogens.